Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors

G protein coupled receptor (GPCR) signalling is mediated by transactivation independent and transactivation dependent pathways. GPCRs transactivate protein tyrosine kinase receptors (PTKRs) and protein serine/threonine kinase receptors (PS/TKR). Since the initial observations of transactivation dependent signalling, there has been an effort to understand the mechanisms behind this phenomena. GPCR signalling has evolved to include biased signalling. Biased signalling, whereby selected ligands can activate the same GPCR that can generate multiple signals, but drive only a unique response. To date, there has been no focus on the ability of biased agonists to activate the PTKR and PS/TKR transactivation pathways differentially. As such, this represents a novel direction for future research. This review will discuss the main mechanisms of GPCR mediated receptor transactivation and the pathways involved in intracellular responses.     

The role of G-protein coupled receptors and associated proteins in receptor tyrosine kinase signal transduction

It is well established that stimulation of G-protein coupled receptors (GPCRs) can activate signalling from receptor tyrosine kinases by a process termed transactivation. Indeed, in recent years, it has become apparent that transactivation is a general phenomenon that has been demonstrated for many unrelated GPCRs and receptor tyrosine kinases. In this case the GPCR/G-protein participation is up-stream of the receptor tyrosine kinase. Substantial research has addressed these findings but meanwhile another mechanism of cross talk has been slowly emerging. For over a decade, a growing body of evidence has demonstrated that numerous growth factors use G-proteins and attendant signalling molecules such as beta-arrestins that participate down-stream of the receptor tyrosine kinase to signal to effectors, such as p42/p44 MAPK. This review highlights this novel mechanism of cross talk between receptor tyrosine kinases and GPCRs, which is distinct from growth factor receptor transactivation by GPCRs.     

Protein tyrosine kinase-mediated pathways in G protein-coupled receptor signaling

Abundant evidence has indicated that protein tyrosine kinases (PTKs) convey signals from G protein-coupled receptors (GPCRs) to regulate cell proliferation, migration, adhesion, and potentialy cellular transformation. Molecular mechanisms by which PTKs regulate such diverse effects in GPCR signaling are not well understood. Recently, an unifying theme has emerged where both growth factors and GPCRs utilize protein tyrosine kinase activity and the highly conserved Ras/MAP kinase pathway to control mitogenic signals. Additionally, PTKs are also involved in the regulation of signal transmission from GPCRs to activation of the JNK/SAPK kinase pathway. Furthermore novel insights in chemokine receptor-activated PTKs and their role in mediating cell functions are discussed in this review.     

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D₃ receptor (D₃R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D₃R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D₃R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D₃R, which can be extended to the crosstalk between other RTKs and GPCRs.     

Bradykinin signalling to MAP kinase: cell-specific connections versus principle mitogenic pathways

Mitogenic signalling pathways from G protein-coupled receptors (GPCRs) to the mitogen-activated protein kinase (MAPK) cascade may involve alpha- or betagamma-subunits of heterotrimeric G proteins, receptor or non-receptor tyrosine kinases, adaptor molecules, phosphoinositide 3-kinases, protein kinase C, and probably other proteins. The majority of models describing the connection of different signalling proteins within a mitogenic pathway are based on experimental data obtained by co- and overexpression of epitope-tagged MAPK together with the respective GPCR and other signalling proteins of interest in transfectable cell lines. Here the link of the bradykinin B2 receptor (B2R) to MAPK in the COS-7 cell expression system is compared with mitogenic signalling pathways of bradykinin in various tumour cell lines. It becomes evident that in natural or tumour cells expressing individual amounts and different isoforms of signalling proteins completely other relations between B2R and MAPK may exist than in COS-7 cells, suggesting a high degree of cellular specificity in mitogenic signalling.     

表皮生长因子诱导人成纤维细胞基因表达谱的变化

深入研究EGF对靶细胞的作用有助于揭示细胞增殖、转化、凋亡以及胚胎发育的分子机理。本文采用cDNA微阵列技术,检测EGF持续作用48小时后人胚肺二倍体成纤维细胞基因表达谱的变化。结果显示:EGF长期作用诱导广泛的基因表达变化,在所检测的4096种人类基因中,有855种发生了变化,这些表达变化的基因参与细胞的能量代谢、生物合成、细胞周期调控及受体酪氨酸激酶和G蛋白耦连受体信号转导等细胞反应,其中最显著的变化趋势是GPCR及其相关蛋白基因表达的下调。     

The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor

Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the "transactivation" of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor-mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. beta(2)-Adrenergic receptor (beta(2)AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the beta(2)AR and the "transactivated" EGFR. beta(2)AR-mediated EGFR phosphorylation and subsequent beta(2)AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. beta(2)AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin-mediated endocytosis, suggesting that signaling downstream of both the EGF-activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery.     

A novel role for phosphatidylinositol 3-kinase beta in signaling from G protein-coupled receptors to Akt

The protein kinase Akt plays a central role in a number of key biological functions including protein synthesis, glucose homeostasis, and the regulation of cell survival or death. The mechanism by which tyrosine kinase growth factor receptors stimulate Akt has been recently defined. In contrast, the mechanism of activation of Akt by other cell surface receptors is much less understood. For G protein-coupled receptors (GPCRs), conflicting data suggest that these receptors stimulate Akt in a cell type-specific manner by a yet to be fully elucidated mechanism. Here, we took advantage of the availability of cells, where Akt activity could not be enhanced by agonists acting on this large family of cell surface receptors, such as NIH 3T3 cells, to investigate the pathway linking GPCRs to Akt. We present evidence that expression of phosphatidylinositol 3-kinase (PI3K) beta is necessary and sufficient to transmit signals from G proteins to Akt in these murine fibroblasts and that the activation of PI3Kbeta may represent the most likely mechanism whereby GPCRs stimulate Akt, as the vast majority of cells do not express PI3Kgamma, a known G protein-sensitive PI3K isoform. Furthermore, available evidence indicates that GPCRs activate Akt by a pathway distinct from that utilized by growth factor receptors, as it involves the tyrosine phosphorylation-independent activation of PI3Kbeta by G protein betagamma dimers.     

Signalling specificity in GPCR-dependent Ca2+ signalling

Cells use signalling networks to translate with high fidelity extracellular signals into specific cellular functions. Signalling networks are often composed of multiple signalling pathways that act in concert to regulate a particular cellular function. In the centre of the networks are the receptors that receive and transduce the signals. A versatile family of receptors that detect a remarkable variety of signals are the G protein-coupled receptors (GPCRs). Virtually all cells express several GPCRs that use the same biochemical machinery to transduce their signals. Considering the specificity and fidelity of signal transduction, a central question in cell signalling is how signalling specificity is achieved, in particular among GPCRs that use the same biochemical machinery. Ca(2+) signalling is particularly suitable to address such questions, since [Ca(2+)](i) can be recorded with excellent spatial and temporal resolutions in living cells and tissues and now in living animals. Ca(2+) is a unique second messenger in that both biochemical and biophysical components form the Ca(2+) signalling complex to regulate its concentration. Both components act in concert to generate repetitive [Ca(2+)](i) oscillations that can be either localized or in the form of global, propagating Ca(2+) waves. Most of the key proteins that form Ca(2+) signalling complexes are known and their activities are reasonably well understood on the biochemical and biophysical levels. We review here the information gained from studying Ca(2+) signalling by GPCRs to gain further understanding of the mechanisms used to generate cellular signalling specificity.     

Transactivation joins multiple tracks to the ERK/MAPK cascade

Many agonists of G-protein-coupled receptors (GPCRs) can stimulate receptor tyrosine kinases and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. A 'transactivation' mechanism, which links these events in one signalling chain, inspired many researchers, but inevitably raised new questions. A 'multi-track' model for GPCR signalling to the ERK/MAPK pathway might resolve some of the puzzles in the transactivation field.     

Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity

G protein-coupled receptors (GPCRs) can stimulate the mitogen-activated protein kinase (MAPK) cascade and thereby induce cellular proliferation like receptor tyrosine kinases (RTKs). Work over the past 5 years has established several models which reduce the links of G(i)-, G(q)-, and G(s)-coupled receptors to MAPK on few principle pathways. They include (i) Ras-dependent activation of MAPK via transactivation of RTKs such as the epidermal growth factor receptor (EGFR), (ii) Ras-independent MAPK activation via protein kinase C (PKC) that converges with the RTK signalling at the level of Raf, and (iii) activation as well as inactivation of MAPK via the cAMP/protein kinase A (PKA) pathway in dependency on the type of Raf. Most of these generalizing hypotheses are founded on experimental data obtained from expression studies and using a limited set of individual receptors. This review will compare these models with pathways to MAPK found for a great variety of peptide hormone and neuropeptide receptor subtypes in various cells. It becomes evident that under endogenous conditions, the transactivation pathway is less dominant as postulated, whereas pathways involving isoforms of PKC and, especially, phosphoinositide 3-kinase (PI-3K) appear to play a more important role as assumed so far. Highly cell-specific and unusual connections of signalling proteins towards MAPK, in particular tumour cells, might provide points of attacks for new therapeutic concepts.     

Ligand-independent and EGF receptor-supported transactivation: lessons from beta2-adrenergic receptor signalling

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) is currently understood to be mediated by matrix metalloproteases (MMPs) and the release of EGF-like ligands. This ligand-mediated process also suggests that downstream of EGFR the signalling in response to GPCR ligands or EGF appears to be indistinguishable. Here we provide evidence that transactivation of EGFR by the beta2-adrenergic receptor (beta2-AR) is independent of MMPs and results in an incomplete downstream signalling involving extracellular signal-activated kinase (ERK) but not PLCgamma1 and Akt. In contrast, beta2-AR has the ability to activate PLCgamma1 when the EGFR is primed either by co-stimulation with EGF or by increased basal activity due to over-expression. In that way but not via the beta2-AR-mediated transactivation the EGFR docking sites pY992 and pY1173 may be generated which are critical for PLCgamma1. This EGFR-supported transactivation is strongly dependent on EGFR tyrosine kinase, c-Src, and the c-Src-specific EGFR tyrosine residue 845 and represents a novel paradigm of EGFR transactivation.     

Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR₁, PAR₂, PAR₃ and PAR₄, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects.In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease.     

Tuberin: a stimulus-regulated tumor suppressor protein controlled by a diverse array of receptor tyrosine kinases and G protein-coupled receptors

Tuberin, a tumor suppressor protein, is involved in various cellular functions including survival, proliferation, and growth. It has emerged as an important effector regulated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Regulation of tuberin by RTKs and GPCRs is highly complex and dependent on the type of receptors and their associated signaling molecules. Apart from Akt, the first kinase recognized to phosphorylate and inactivate tuberin upon growth factor stimulation, an increasing number of kinases upstream of tuberin have been identified. Furthermore, recruitment of different scaffolding adaptor components to the activated receptors appears to play an important role in the regulation of tuberin activity. More recently, the differential regulation of tuberin by various G protein family members have also been intensively studied, it appears that G proteins can both facilitate (e.g., G(i/o)) as well as inhibit (e.g., G(q)) tuberin phosphorylation. In the present review, we attempt to summarize our emerging understandings of the roles of RTKs, GPCRs, and their cross-talk on the regulation of tuberin.     

Emerging roles for β-arrestin-1 in the control of the pancreatic β-cell function and mass: New therapeutic strategies and consequences for drug screening

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase in β-cell mass, decreased β-cell survival and impaired glucose-dependent insulin release. Pancreatic β-cell proliferation, survival and secretion are thought to be regulated by signalling pathways linked to G-protein coupled receptors (GPCRs), such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors. β-arrestin-1 serves as a multifunctional adaptor protein that mediates receptor desensitization, receptor internalization, and links GPCRs to downstream pathways such as tyrosine kinase Src, ERK1/2 or Akt/PKB. Importantly, recent studies found that β-arrestin-1 mediates GLP-1 signalling to insulin secretion, GLP-1 antiapoptotic effect by phosphorylating the proapoptotic protein Bad through ERK1/2 activation, and PACAP potentiation of glucose-induced long-lasting ERK1/2 activation controlling IRS-2 expression. Together, these novel findings reveal an important functional role for β-arrestin-1 in the regulation of insulin secretion and β-cell survival by GPCRs.     

EGF receptor transactivation is obligatory for protein synthesis stimulation by G protein-coupled receptors

The epidermalgrowth factor receptor (EGFR) was recently identified as a signaltransducer of G protein-coupled receptors (GPCRs). In this study, wehave examined the contribution of EGFR transactivation to thegrowth-promoting effect of GPCRs on vascular smooth muscle cells.Activation of the G_q-coupled ANG II receptor orG_i-coupled lysophosphatidic acid receptor resulted inincreased tyrosine phosphorylation and activation of EGFR. Specificinhibition of EGFR kinase activity by tyrphostin AG-1478 or expressionof a dominant-negative EGFR mutant abolished this response.Importantly, inhibition of EGFR function strongly attenuated the globalstimulation of protein synthesis by GPCR agonists in vitro in culturedaortic smooth muscle cells and in vivo in the rat aorta and in small resistance arteries. The growth inhibition was associated with a markedreduction of extracellular signal-regulated kinase and phosphoinositide3-kinase pathway activity and the resulting suppression of eukaryotictranslation initiation factor 4E and 4E binding protein 1 phosphorylation. Our results demonstrate that EGFR transactivation is aphysiologically relevant action of GPCRs linked to translational control and protein synthesis.

     

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.     

Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins

G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.     

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.     

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.